Functional Investigation of the Plant-Specific Long Coiled-Coil Proteins PAMP-INDUCED COILED-COIL (PICC) and PICC-LIKE (PICL) in <em>Arabidopsis thaliana</em>

<div><p>We have identified and characterized two Arabidopsis long coiled-coil proteins PAMP-INDUCED COILED-COIL (PICC) and PICC-LIKE (PICL). PICC (147 kDa) and PICL (87 kDa) are paralogs that consist predominantly of a long coiled-coil domain (expanded in PICC), with a predicted transmembrane domain at the immediate C-terminus. Orthologs of PICC and PICL were found exclusively in vascular plants. PICC and PICL GFP fusion proteins are anchored to the cytoplasmic surface of the endoplasmic reticulum (ER) membrane by a C-terminal transmembrane domain and a short tail domain, via a tail-anchoring mechanism. T-DNA-insertion mutants of <i>PICC</i> and <i>PICL</i> as well as the double mutant show an increased sensitivity to the plant abiotic stress hormone abscisic acid (ABA) in a post-germination growth response. <i>PICC,</i> but not <i>PICL</i> gene expression is induced by the bacterial pathogen-associated molecular pattern (PAMP) flg22. T-DNA insertion alleles of <i>PICC,</i> but not <i>PICL,</i> show increased susceptibility to the non-virulent strain <i>P. syringae</i> pv. <i>tomato</i> DC3000 <i>hrcC</i>, but not to the virulent strain <i>P. syringae</i> pv. <i>tomato</i> DC3000. This suggests that <i>PICC</i> mutants are compromised in PAMP-triggered immunity (PTI). The data presented here provide first evidence for the involvement of a plant long coiled-coil protein in a plant defense response.</p> </div

PICC and PICL promoters have partially overlapping patterns of activity.

<p>(<b>A</b>) β-glucuronidase staining indicating PICC promoter activity in the vasculature of cotyledons, roots, young and mature leaves (I, II and III), in the hydathodes (arrows in III and IV), in the trichomes (IV and inset in IV), in the vasculature of sepals and petals (V and VI), in the filaments of the anther (VI), in the stem and at the nodes (VII) and in the abscission zone of flowers and siliques (arrows in VIII and IX). (<b>B</b>) β-glucuronidase staining indicating PICL promoter activity in the vasculature of cotyledons and young leaves (I, II and III), in the vasculature of hypocotyls and roots (I), in the hydathodes (arrow in IV) and at the nodes (V). No activity was visible in the buds, flowers (VI) and siliques (not shown) of the inflorescence.</p

PICL and PICC are associated with the ER via their C-terminal transmembrane domain.

<p>(<b>A</b>) N-terminally tagged GFP-fusion proteins used in this study. Amino acid sequence of the transmembrane domain and the C-terminal tail are shown in blue and red letters, respectively. Numbers indicate amino acid positions. Drawings are not to scale. (<b>B</b>) Confocal images showing localization of the fusion proteins indicated on the left in <i>N. benthamiana</i> and Arabidopsis leaf epidermal cells. Cytoplasmic localization of unfused GFP (“Free GFP”) in <i>N. benthamiana</i> and Arabidopsis are shown as controls (bottom right). Scale  = 10 µm.</p

<i>picc-1</i> and <i>picc-1;picl-1</i> Arabidopsis plants are more susceptible to <i>hrcC</i>.

<p>Levels of type III secretion deficient <i>hrcC</i> and wild-type <i>Pst</i>DC3000 four days after infiltration into leaves of the indicated plants. Values represent average of three replicates. Error bars represent one standard deviation. Increased growth of <i>hrcC</i> in <i>picc-1</i> and/or <i>picc-1;picl-1</i> relative to WT was observed in 6 out of 7 biological replicates. CFU, Colony Forming Units.</p

Time course of <i>PICC</i> induction.

<p>4-week-old WT Col-0 plants were infiltrated with 1 µM flg22 (<b>A</b>), or 2×10⁸ CFU ml⁻¹ type III secretion deficient <i>hrcC</i> (<b>B</b>). <i>PICC</i> steady state mRNA levels were quantified by real-time PCR at times indicated. Transcript levels were normalized to <i>ACTIN</i> levels from the same sample. Values are given in arbitrary units with the value in 1 h flg22 treated sample set to 1. Each value is represented as the average of three biological replicates for treatment with flg22 (A) and <i>hrcC</i> (B). Error bars represent one standard deviation. Double (**P<0.01) and single (* P<0.05) asterisks indicate statistically significant difference in values compared to mock treated samples at the corresponding time point.</p

<i>picl-1</i>, <i>picc-1;picl-1, picc-1</i>, and <i>picc-2</i> are hypersensitive to ABA at the post-germination growth stage.

<p>WT, <i>picl-1, picc-1;picl-1, picc-1,</i> and <i>picc-2</i> were grown on MS plates containing different concentrations of ABA. Post-germination growth efficiency was determined as percentage of green and expanded cotyledons at 10 days after stratification. Values represent average of three replicates, where number of seeds  = 54 in each replicate. Error bars represent one standard deviation. On plates containing 0 µM ABA, WT and mutants had 100% post germination growth efficiency. Similar results were obtained in three out of four biological replicates.</p

PICC expression is induced by flg22.

<p>10-day-old liquid-grown seedlings were treated with water or flg22. <i>PICC</i> (<b>A</b>) <i>and PICL</i> (<b>B</b>) steady-state mRNA levels were quantified by real-time RT-PCR at times indicated. (<b>C</b>) <i>MYB51,</i> a known flg22-induced gene, was used as a positive control. Transcript levels were normalized to <i>ACTIN</i> measured in the same samples. Values are given in arbitrary units with expression in 2 h flg22 treated samples set to 1. Each value is represented as the average of two biological replicates. Error bars represent one standard deviation. Double asterisks (**) indicate statistically significant difference in values compared to mock treated samples at the corresponding time point (P<0.01).</p

PICL and PICC N-termini face the cytoplasm.

<p>Immunoblot analysis using GFP antibody. Microsomal preparations were treated with and without Proteinase K. GFP-CXN and CXN-PAGFP were used as controls. In the microsome fraction containing GFP-CXN, GFP is protected from proteinase K treatment, whereas GFP of CXN-PAGFP is susceptible to proteinase K digestion. GFP of GFP-TDF^PICL and GFP-TDF^PICC are hydrolyzed indicating exposure to Proteinase K. At the given concentration of proteinase K (sufficient to completely hydrolyze GFP in GFP-TDF^PICL and GFP-TDF^PICC), a small amount of PAGFP remains undigested (second column of CXN-PAGFP). Microsomal membranes were solubilized by the detergent Triton X-100. Numbers on the left indicate approximate molecular mass in kilodaltons.</p

Protein structure of PICC and PICL, and phylogenetic tree of PICC, PICL and their orthologs in vascular plants.

<p>(<b>A</b>) Putative protein structure of PICC and PICL showing coiled-coil and transmembrane domains. (<b>B</b>) Graphical representation of the maximum-likehood phylogenetic tree of PICC, PICL and their orthologs. This phylogenetic tree is based on the multiple sequence alignment shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057283#pone.0057283.s001" target="_blank">Figure S1B</a>. Branch support values are indicated at the nodes as calculated by the PhyML program using default parameters. Os, <i>Oryza sativa</i>; Pt, <i>Populus trichocarpa;</i> Rc, <i>Ricinus communis;</i> Sb, <i>Sorghum bicolor;</i> Vv, <i>Vitis vinifera.</i></p

PICC forms homodimers.

<p>β-galactosidase activity as a reporter for interaction in a membrane yeast two-hybrid (split-ubiquitin) assay. PICC shows self-interaction as indicated by increased β-galactosidase activity in yeast containing the constructs Cub-PICC and NubG-PICC. β-galactosidase activity in yeast transformed with combinations of Cub-PICL or Cub-PICC with either the empty vector NubG or the unrelated gene Alg5-NubG were used as negative controls. Combinations of Cub-PICL or Cub-PICC with Alg5 fused to wild-type Nub (Alg5-NubI) were used as positive controls. a.u., arbitrary units. Mean values and standard deviation from 3 samples are shown.</p